FREE (UNESTERIFIED) FATTY ACIDS

1. Occurrence and Biochemistry

Free or unesterified fatty acids are ubiquitous if minor components of all
living tissues. In animals, much of the dietary lipid is hydrolysed to
free acids before it is absorbed and utilized for lipid synthesis.
Intact lipids in tissues can be hydrolysed to free acids by a variety
of lipolytic enzymes (e.g. lipoprotein lipase, hormone-sensitive lipase,
phospholipase A), before being metabolized in various ways including oxidation,
desaturation, elongation or re-esterification. As free acids can interact with a wide range of enzyme systems in
both specific and non-specific ways,
they must be rapidly sequestered in tissues by various means to ensure that their activities are closely regulated.

Monomeric
fatty acids in the free state have very low solubilities in aqueous media.
In serum, they are transported between tissues bound to the protein albumin, which has up to six strong binding sites and
a large number of weak binding sites where non-polar interactions are possible between the fatty acid hydrocarbon chains and
uncharged amino acid side chains.
In this way, the concentration of a long-chain fatty acid in serum
can be increased by as much as 500 times above its normal maximum.
However, the bound fatty acids can diffuse into the aqueous phase,
where they are rapidly taken up into the outer leaflet of the
plasma membrane by non-enzymatic mechanisms. It is then possible that fatty acids cross the membrane simply and
rapidly by a biophysical process, i.e. by ‘flip-flop’.

There is also evidence that specific transporter proteins
may be involved in part to activate by formation of
acyl-coA prior to further esterification, but also to ensure vectorial transport so that
specific fatty acids are directed towards particular purposes.
Certainly within the cell, a family of fatty acid binding or transport proteins has essential functions in fatty acid
trafficking pathways and in fatty acid activation, many of which are specific to particular tissues.
These include uptake of dietary lipids in the intestine,
targeting of fatty acids in the liver to either catabolic or anabolic pathways,
regulation of storage in adipose tissue, targeting to β-oxidation pathways in muscle,
and maintenance of phospholipid compositions in neural tissues.
It appears that cells have several overlapping mechanisms that ensure sufficient uptake and directed intracellular
movement of the fatty acids required for their physiological functions.

Apart from their obvious role as a source of energy (see our web page on
acylcarnitines, for example), unesterified fatty acids can act as second messengers
required for the translation of external signals, as they can be produced rapidly as a consequence of the
binding of specific agonists to plasma membrane receptors. In this way, they can substitute for the second messengers
of the inositide pathways. Fatty acids are effective also in operating at specific intracellular locations reversibly
to amplify or otherwise modify signals. For example, they influence the activities of protein kinases, phospholipases,
G-proteins, adenylate and guanylate cyclases, and many other metabolic processes.
Part of the action of fatty acids may occur indirectly via metabolism of arachidonic acid to eicosanoids.
On the other hand, there is much evidence that fatty acids
per se are messengers that mediate the responses of the cell to extracellular signals.
Many of these reactions are specific to particular fatty acids. For example, polyunsaturated fatty acids,
including docosahexaenoic and arachidonic acids, bind to the retinoid X receptor
and induce activation. Some related processes appear to occur in plants.

In addition in animal tissues, long-chain polyunsaturated fatty acids
are involved in regulating gene expression,
mainly targeting genes that encode proteins with roles in fatty acid transport or metabolism.
In this respect, (n-3) fatty acids are more potent than (n-6) fatty acids.
Straight-chain saturated and monoenoic fatty acids do not appear to be involved in the process,
but surprisingly poly-methyl-branched fatty acids, such as
phytanic and pristanic, may have a function of this kind.
In some circumstances, both the free acids
per se and their coenzyme A esters may be involved.
Fatty acid-binding proteins bind long-chain fatty acids with high affinity in the cytoplasm and transport them to nuclei,
which they enter via the nuclear pores,
where they are able to form complexes with nuclear receptors enabling them to regulate receptor activation.

The
mechanisms by which modulation of gene transcription occurs are only
partially resolved, and this is the subject of considerable research effort,
especially with respect to the family of transcription factors, i.e. peroxisome
proliferator-activated receptors (PPARs), in the nuclei of cells.
The effects can be highly specific, different fatty acids binding to or activating different types of PPAR,
although the PPARα and hepatocyte nuclear factor 4α (HNF4α) are especially important.
In particular, polyunsaturated fatty acids may exert beneficial effects by up-regulating the expression of genes encoding
enzymes involved in oxidation of fatty acids,
while at the same time down-regulating genes for enzymes involved in lipid synthesis.
They also influence glucose metabolism. As a result, unesterified fatty acids may mitigate the undesirable symptoms of
the metabolic syndrome and may even reduce the risk of heart disease.
In contrast, abnormal PPAR activation can be a factor in the lipotoxicity observed with obesity, insulin resistance,
type 2 diabetes and hyperlipidemia. Abnormally elevated levels of non-esterified fatty acids in plasma, for example,
are associated with the pathologies of these disease states.
Also, they increase greatly the amounts of key bioactive lipids in tissues such as the pancreas,
skeletal muscle and adipose tissue.

However, some of the mediator effects appear to be independent of PPARs
and are characterized by involvement with cell surface receptors instead.
Thus, multiple G-protein-coupled receptors for free fatty acids have been identified
that function on the cell surface and have important roles in nutrition regulation.
Some of these are activated by short-chain and others by medium- and long-chain free fatty acids.
The former are expressed preferentially in pancreatic β-cells and mediate insulin secretion.
In certain intestinal cells, there are believed to be such ‘fatty acid sensing molecules’,
which recognise the presence of free long-chain acids in digesta.
These stimulate the release of the hormone cholecystokinin,
which in turn causes the gall-bladder to contract and release bile to aid digestion.
In effect, free fatty acids act as nutrient sensors to regulate energy homeostasis.

Similarly, in bacteria, it has been demonstrated that the bacterial fatty acid transport and trafficking
system leads to fatty acid-responsive regulation of gene expression.

Free fatty acids have potent antimicrobial, antiviral and antifungal properties,
and they exert such effects in some living systems, especially the skin and mucosa of the lung.
As they are powerful detergents and will inhibit very many enzymes systems in a non-specific manner,
it is not clear whether the biocidal properties are also non-specific.
Unsaturated fatty acids seem to have the greatest effects,
but this may be because they can insert more readily into membranes.
At high concentrations in vitro, free fatty acids are known to perturb membrane structures,
but cytotoxic effects come into play before this can become relevant.

2. Analysis

Accurate measurement of free fatty acids concentrations in
plasma and tissues can be a useful measure of metabolic status. Unfortunately,
it is very easy to generate free acids artefactually by faulty storage or
extraction. Lipases can continue to function slowly in some tissues, even at
–20°C, and the process will accelerate if tissues are allowed to thaw prior to
extraction. In one important series of experiments, it was shown that if animal tissues were
pulverized and extracted at –70°C, very low levels only of free
fatty acids were detected in comparison to more conventional
procedures [Kramer, J.K.G. and Hulan, H.W. J. Lipid Res., 19,
103-106 (1978)]. The high concentrations reported occasionally in the literature are
obviously impossible in living tissues and are due to inappropriate sample
handling.

Following extraction of lipids from tissues by a suitable procedure, a free
fatty acid fraction can be isolated by thin-layer chromatography or by
solid-phase extraction chromatography on a bonded amine phase. This
can be methylated and analysed by gas chromatography with an internal
standard.

Thormar, H. and Hilmarsson, H. The role of microbicidal lipids in host defense
against pathogens and their potential as therapeutic agents.Chem. Phys. Lipids, 150, 1-11 (2007)
(DOI: 10.1016/j.chemphyslip.2007.06.220).